The ability to produce recombinant human proteins has led to major advances in human health care and remains an active area of drug discovery. Many therapeutic proteins require the cotranslational addition of glycans to specific asparagine residues (N-glycosylation) of the protein to ensure proper structure-function activity and subsequent stability in human serum. For therapeutic use in humans, glycoproteins require human-like N-glycosylation. Mammalian cell lines (Chinese hamster ovary (CHO) cells as well as human retinal cells) which can mimic human-like glycoprotein processing have several drawbacks including low protein titers, long fermentation times, heterogeneous products, and ongoing viral containment issues. Thus, the use of yeast and filamentous fungal expression systems having more economical processing, fewer safety obstacles and producing more robust heterologous protein yields have been heavily researched as host cells for human therapeutics.
In yeast and filamentous fungus, glycoproteins are produced having oligosaccharides which are different from those of mammalian-derived glycoproteins. Specifically in yeast, outer chain oligosaccharides are hypermannosylated consisting of 30-150 mannose residues (Kukuruzinska et al., 1987, Annu. Rev. Biochem. 56: 915-944). Moreover, mannosylphosphate is often transferred to both the core and outer sugar chains of glycoproteins produced in yeast (Ballou, 1990, Methods Enzymol. 185: 440-470). Of most consequence, is that these mannosylphosphorylated glycans from glycoproteins produced in the yeast, Saccharomyces cerevisiae, have been shown to illicit an immune response in rabbits (Rosenfeld and Ballou, 1974, JBC, 249: 2319-2321). Thus, the elimination of mannosylphosphorylation in yeast and filamentous fungi is essential for the production of non-immunogenic therapeutic glycoproteins.
In S. cerevisiae there are at least two genes which participate in the transfer of mannosylphosphate. The two genes, MNN4 and MNN6 have been cloned, and analyses of the gene products suggest they function in the transfer of mannosylphosphate (for review see Jigami and Odani, 1999, Biochim. Biophys. Acta, 1426: 333-345). MNN6 encodes a type II membrane protein homologous to the Kre2p/Mnt1p family of proteins which has been characterized as Golgi α-1,2-mannosyl-transferases involved in O-mannosylation and N-glycosylation (Lussier et al., 1997, JBC, 272: 15527-15531). The Δmnn6 mutant does not show a defect in the mannosylphosphorylation of the core glycans in vivo, but exhibits a decrease in mannosylphosphate transferase activity in vitro (Wang et al., 1997, JBC, 272: 18117-18124). Mnn4p is also a putative type II membrane protein which is 33% identical to the S. cerevisiae Yjr061p (Odani et al., 1996, Glycobiology,6: 805-810; Hunter and Plowman, 1997, Trends in Biochem. Sci., 22:18-22). Both the Δmnn6 and Δmnn4 mutants decrease the transfer of mannosylphosphate. However, the Δmnn6Δmnn4 double mutant does not further reduce this activity. These observations suggest the presence of additional mannosyltransferases that add mannosylphosphate to the core glycans.
Thus, despite the reduction of mannosylphosphorylation in S. cerevisiae with the disruption of MNN4, MNN6 or both in combination, there is no evidence that complete elimination of mannosylphosphate transferase activity is possible. Other genes which affect the mannosylphosphate levels have been identified in S. cerevisiae. These genes include PMR1, VRG4, MNN2 and MNN5. PMR1 encodes a Golgi-localized Ca2+/Mn2+-ATPase required for the normal function of the Golgi apparatus (Antebi and Fink, 1992, Mol. Biol. Cell, 3: 633-654); Vrg4p is involved in nucleotide-sugar transport in the Golgi (Dean et al., 1997, JBC, 272: 31908-31914), and Mnn2p and Mnn5p are α1,2-mannosyltransferases responsible for the initiation of branching in the outer chain of N-linked glycans (Rayner and Munro, 1998, JBC, 273: 23836-23843). For all four proteins, the reduction in mannosylphosphate groups attached to N-linked glycans seems to be a consequence of Golgi malfunction or a reduction in size of the N-linked glycans rather than a specific defect in the transfer activity of the mannosylphosphate groups.
Proteins expressed in the methylotrophic yeast, Pichia pastoris contain mannosylphosphorylated glycans (Miele, et al., 1997, Biotech. Appl Biochem., 2: 79-83). Miura et al. reported the identification of the PNO1 (Phosphorylmannosylation of N-linked Oligosaccharides) gene which upon disruption confers an attenuation of mannosylphosphorylation on glycoproteins (WO 01/88143; Miura et al., 2004, Gene, 324: 129-137). The PNO1 gene encodes for a protein involved in the transfer of mannosylphosphate to glycans in P. pastoris. Its specific function, however, is unknown. As mentioned, the Δpno1 mutant decreases but does not abolish mannosylphosphorylation on N-glycans relative to a P. pastoris strain having wild-type Pno1p.
Currently, no methods exist to eliminate mannosylphosphorylation on glycoproteins produced in fungal hosts. A residual amount of mannosylphosphorylation on glycoproteins may still be immunogenic and, thus, is undesirable for use as human therapeutics.
What is needed, therefore, is an expression system based on yeast or filamentous fungi that produces glycoproteins which are essentially free of mannosylphosphorylated glycans.